This invention relates to a method of reducing the pyrogenicity, hepatitis infectivity and clotting activation of plasma, plasma derivatives and products thereof.
In the development of new products from human plasma, at least three major problems are always present. These are contamination with pyrogens (endotoxins), transmission of viral hepatitis, and activation of the coagulation enzymes.
Pyrogens are lipopolysaccharides (LPS) derived from the outer cell wall of gram-negative bacteria. They are toxic materials which are also known as endotoxins to distinguish them from toxic substances synthesized and excreted by the intact bacterium. Pyrogens have numerous biologic activities which include the production of fever, activation of clotting mechanisms and induction of shock. Consequently, it is essential that pyrogenic substances be removed and that the causative bacteria be rendered innocous by sterilization or other such treatment of the final plasma product.
Prior methods for such inactivation or destruction of pyrogens comprise extensive treatment with heat, acid or alkali, filtration of insoluble pyrogens, or removal by adsorption with gels, ion-exchange resins and various other such adsorbent materials. Most of these methods are burdensome, time consuming, or destructive of the protein due to the rigorousness of the treatment.
Further background on the properties and effects of pyrogens can be had by reference to a paper by Elizabeth Work entitled "Production, Chemistry and Properties of Bacterial Pyrogens and Endotoxins" in "Pyrogens and Fever", Ciba Foundation Symposium, 1971, pp. 23-47, edited by Wolstenholme and Birch, published by Churchill Livingstone; and a paper by D. C. Morrison and R. J. Ulevitch entitled "The Effects of Bacterial Endotoxins on Host Mediation Systems" in Amer. J. Pathol. 93 (2), 527-601 (1978).
It is well-known that plasma and products made from plasma may transmit hepatitis. Until recently, interest has focused primarily on hepatitis B antigen (HB.sub.s Ag) as the offending agent and attempts at eliminating this agent have led to widespread screening of all plasma used in transfusing by a variety of laboratory procedures. While such laboratory screening has apparently decreased the incidence of hepatitis B in patients receiving whole blood transfusions, there has not been significant improvement in the incidence of the disease transmitted from plasma products. Attempts to remove the virus by various adsorption procedures or precipitation techniques, e.g. with polyethylene glycol, have not proven to eliminate infectivity. There is some evidence that the combination of ultraviolet light and .beta.-propionolactone may be helpful in inactivating the virus in certain plasma products. However, there is some apprehension that .beta.-propionolactone has carcinogenic properties.
While the development of screening tests for hepatitis B has been of limited value in reducing transmission of the disease, the identification of this virus (as well as the hepatitis A virus) has led to the recognition of a third virus which is apparently responsible for the majority of cases of hepatitis transmitted by blood plasma derivatives. This virus is referred to as "non-A, non-B hepatitis". Tests for this virus are not yet commercially available for widespread screening. This virus closely resembles hepatitis B virus but is antigenically distinguishable. Both hepatitis B virus and non-A, non-B hepatitis virus appear to have similar structural characteristics and exist as particles containing a DNA core and a lipoprotein membrane.
Attempts to prevent activation of clotting enzymes have centered around the addition of classic anticoagulating chemicals such as citrate, EDTA and heparin. While such measures are partially effective, none of these anticoagulants are effective at the early stage of the clotting sequence which is triggered by activation of Factor XII. Activation of clotting factors may still occur because coagulation may be instigated by the presence of phospholipids from blood platelets. Virtually all blood fractions collected today contain thromboplastic-like substances or phospholipids from platelets and platelet derivatives which activate coagulation. Administration of therapeutic blood fractions which contain these phospholipid particles may be potentially dangerous because of their ability to induce unwanted intravascular coagulation.